Quinazolinone compounds in combined modalities for improved cancer treatment

ABSTRACT

The present invention provides compositions and methods for improving the effectiveness of anti-tumor treatments. The compositions of the present invention comprise quinazolinones, specifically halofuginone. In currently preferred embodiments the compositions and methods of the present invention improve the effectiveness of radiation therapy and chemotherapy, and concomitantly alleviate or prevent the damage induced by radiation therapy.

FIELD OF THE INVENTION

The present invention relates to the field of cancer treatment,specifically to the synergistic effects obtained by the administrationof quinazolinone derivatives, particularly halofuginone, in conjunctionwith additional anti tumor therapies.

BACKGROUND OF THE INVENTION

Fibrosis

Clinical conditions and disorders associated with primary or secondaryfibrosis are characterized by excessive production of connective tissue,resulting in destruction of normal tissue architecture and function.Fibrosis results from diverse modes of trauma including burns, surgery,infection, alcohol consumption and exposure to toxins.

Acute fibrosis is also a common adverse effect associated with cancertherapy, including radiation and chemotherapy treatments.

Radiation and fibrosis

Radiation fibrosis is an extremely severe adverse effect of ionizingradiation employed in therapy of various cancerous conditions. Fibrosismay develop as a sequel of the necessary radiotherapy and the accidentaloverexposures associated with the therapy. As of today, preventive orcurative treatment for radiation fibrosis is not available.

Fibrosis disorders following radiation have been described in almost anytissue, including skin, lung, heart, liver and kidneys, and have shownto cause acute complications (such as bowel obstruction, severe lunginjury, etc.).

Medical treatments used to overcome such acute complications resultingfrom radiation fibrosis were not shown to have beneficial effects. Themost common method used is surgery, which is rarely successful,generally requires repeated operations, and is accompanied with poorrecovery.

The clinical conditions and disorders related to radiation fibrosis arecharacterized by excessive production of connective tissue, resulting inthe destruction of normal tissue architecture and function.

Although radiation fibrosis has been reported for many years inhistopathological studies, the mechanisms of its initiation and chronicextension still remain to be resolved. Fibrosis is in fact a dynamicprocess, characterized by constant remodeling and long term fibroblastactivation. In normal wound healing, fibroblasts are transientlyactivated into myofibroblasts to proliferate and deposit the collagenmatrix. Feedback mechanisms then occur to down regulate cellularactivities, and it has been proposed that myofibroblasts becometerminally differentiated and finally disappear due to apoptosis. On thecontrary, in fibrosis, the feedback regulations are not observed, andchronic, long term myofibroblast activation is sustained. One possibleorigin of the chronic cellular activation could be an abnormalproduction of stimulating factors such as cytokines and growth factors.

Recently, a new concept was proposed regarding the initiation ofradiation damage, suggesting that a cascade of cytokines initiatedimmediately after irradiation persists for long periods of time andleads to the development of late damage.

Chemotherarpy and Fibrosis

Several cytotoxic agents commonly used in chemotherapy are known toinduce fibrosis in different organs. One of the most widely reportedagents is Bleomycin, which is known to induce lung fibrosis. Otheragents associated with high number of fibrosis incidence includebusulfan, carmustine (BCNU), and mitomycin-C.

Bleomycin is reported to induce pulmonary fibrosis in approximately 10%to 30% of treated patients, with death of 1% to 2% of patientsassociated with pulmonary fibrosis (Wesselius L., J. Comp. Ther. 1999:25(5):272-277).

Intra-abdominal and retroperitoneal fibrosis have been described assecondary to intraperitoneal (IP) administration of severalchemotherapeutic agents, including carboplatin, mitoxantrone and thecombination of 5-fluorouracil and cisplatin (Fata et. al., Cancer 2000,June 1:88(11):2447-51).

Adriamycin, administered either in conventional or liposomalformulations, is known to induce fibrotic encapsulation of tumors thatdecreases the concentrations of the drug in the tumor, leading toreduced efficacy of the chemotherapy.

Halofuginone

U.S. Pat. No. 3,320,124 disclosed and claimed a method for treatingcoccidiosis with quinazolinone derivatives. Halofuginone, otherwiseknown as7-bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl]-4(3H)-quinazolinone(one of the quinazolinone derivatives), was first described and claimedin said patent to American Cyanamid, and was the preferred compoundtaught by said patent and the one commercialized from among thederivatives described and claimed therein. Subsequent U.S. Pat. Nos.4,824,847; 4,855,299; 4,861,758 and 5,215,993 all relate to thecoccidiocidal properties of Halofuginone.

More recently, some of the inventors of the present invention (U.S. Pat.No. 5,449,678 to Pines et. al) disclosed that these quinazolinonederivatives are unexpectedly useful for the treatment of a fibroticcondition. That disclosure provides compositions of a specific inhibitorcomprising a therapeutically effective amount of a compound having thegeneral formula I:

wherein: n=1-2R₁ is at each occurrence independently selected from the groupconsisting of a member of the group consisting of hydrogen, halogen,nitro, benzo, lower alkyl, phenyl and lower alkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy; andR₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof.

The '678 patent discloses that these compounds are effective in thetreatment of fibrotic conditions such as scleroderma and graft versushost disease (GVHD). Of this group of compounds, halofuginone has beenfound to be particularly effective.

Some of the inventors of the present invention have further disclosed inU.S. Pat. No. 5,891,879 to Nagler et al. that these compounds areeffective in treating restenosis. Both conditions, namely fibrosis andrestenosis are associated with excessive collagen deposition, which canbe inhibited by halofuginone. Restenosis is characterized by smoothmuscle cell proliferation and extracellular matrix accumulation withinthe lumen of affected blood vessels in response to a vascular injury(Choi et al., Arch. Surg., 1995, 130:257-261). One characteristic ofsuch smooth muscle cell proliferation is a phenotypic alteration, fromthe normal contractile phenotype to a synthetic one. Type I collagen hasbeen shown to support such a phenotypic alteration, which can be blockedby halofuginone (Choi et al., Arch. Surg., 130:257-261, 1995; U.S. Pat.No. 5,449,678).

Notably, halofuginone inhibits collagen synthesis by fibroblasts invitro, however, it promotes wound healing in vivo (WO 01/17531 to Nagleret. al). Thus, the exact behavior of halofuginone in vivo cannot alwaysbe accurately predicted from in vitro studies.

In addition, pharmaceutical compositions comprising quinazolinone,including halofuginone, have been disclosed and claimed as effective fortreating malignancies (U.S. Pat. No. 6,028,075 to Pines et. al) as wellas for prevention of neovascularization (U.S. Pat. No. 6,090,814 toNagler et. al).

The ability of halofuginone, or other related quinazolinone derivatives,to enhance the efficacy of known anti-tumor treatments, particularlyradiation or chemotherapy, was neither taught or suggested in thebackground art. Such enhancement may reduce the dose required forsuccessful anti-tumor treatment, leading to a reduction in the undesiredadverse effects, including fibrosis.

SUMMARY OF THE INVENTION

It is now disclosed that pharmaceutical compositions comprisingquinazolinone derivatives, specifically halofuginone, can unexpectedlyimprove the effectiveness of anti tumor treatments, such as radiationand chemotherapy. The present invention further proposes that thesynergistic effect of quinazolinone is mediated by increasing thesensitivity of tumor cells to the ionizing radiation or to thechemotherapy treatment.

According to one aspect the present invention provides a method forincreasing the effectiveness of anti-tumor treatments, the methodcomprising the step of co-administering to a subject in need thereof apharmaceutical composition comprising as an active ingredient aquinazolinone derivative compound having the general formula I:

wherein: n=1-2R₁ at each occurrence is independently a member of the group consistingof hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and loweralkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy;R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof,and at least one additional anti tumor treatment.

According to one currently preferred embodiment, the quinazolinonederivative is halofuginone.

According to one embodiment, the at least one additional anti tumortreatment administered in combination with the quinazolinonecompositions of the present invention is selected from the groupconsisting of radiation therapy, chemotherapy, immunotherapy, hormonaltherapy and genetic therapy.

According to one currently preferred embodiment the anti tumor treatmentis selected from the group consisting of radiation or chemotherapy.

According to one embodiment, the chemotherapeutic agent is selected fromthe group consisting of topoisomerase inhibitors, spindle poison vincas:vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel;alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide,melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil,cytarabine, gemcitabin; podophyllotoxins: etoposide, irinotecan,topotecan, dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin,mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin,idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin;interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide,megestrol acetate.

According to one embodiment the co-treatment of the present invention isperformed by separate administrations of each of the treatments, namelythe administration of quinazolinone compositions and the administrationof at least one additional anti tumor treatment.

According to another embodiment, the administration of the quinazolinonecomposition is essentially at the same time as the administration of theadditional anti tumor treatment.

According to another embodiment when the additional anti tumor treatmentis chemotherapy, co-administration of the two agents, whether as asingle combined composition or in separate compositions, is also shownto act synergistically.

According to another embodiment the present invention provides a methodfor increasing the effectiveness of additional anti-tumor treatments, bypre-administering quinazolinone derivative compounds having the generalformula I:

wherein: n=1-2R₁ at each occurrence is independently a member of the group consistingof hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and loweralkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy;R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof,followed by the administration of at least one additional anti-tumortreatment.

Treatment with quinazolinones according to the present invention can beparticularly effective and beneficial when administered prior to theadministration of an additional anti-tumor chemotherapeutic agent or totreatment with radiation therapy. This advantage is attained by the useof halofuginone to synchronize the cells, thereby rendering them moresusceptible to the subsequent anti-tumor treatment.

According to another aspect the present invention provides a combinedcomposition for increasing the effectiveness of anti tumor treatments,comprising a quinazolinone derivative compound having the generalformula 1:

wherein: n=1-2R₁ is a at each occurrence independently a member of the groupconsisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl andlower alkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy;R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof,further comprising at least one additional anti tumor agent.

According to one currently preferred embodiment the combined compositioncomprises halofuginone.

According to another embodiment, the at least one additional anti tumoragent present in combination with the quinazolinone in the compositionsof the present invention is selected from the group consisting oftopoisomerase inhibitors, spindle poison vincas: vinblastine,vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylatingagents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan,ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine,gemcitabin; podophyllotoxins: etoposide, irinotecan, topotecan,dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin;nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin,daunorubicin; inorganic ions: cisplatin, carboplatin; interferon,asparaginase; hormones: tamoxifen, leuprolide, flutamide, megestrolacetate.

According to yet another aspect the present invention provides the useof a quinazolinone derivative having the general formula I:

wherein: n=1-2R₁ at each occurrence is independently a member of the group consistingof hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and loweralkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy; andR₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof,in preparation of a medicament for treating a tumor in combinationtherapy with at least one additional anti tumor treatment, therebyimproving the effectiveness of the anti tumor treatment.

According to one currently preferred embodiment the quinazolinonederivative used in combined therapy is halofuginone.

According to one embodiment, the combined therapy comprises anadditional known anti tumor treatment selected from the group consistingof radiation therapy, chemotherapy, immunotherapy, hormonal therapy andgenetic therapy.

According to one currently preferred embodiment the combined therapycomprises an additional anti tumor treatment selected from the groupconsisting of radiation or chemotherapy.

According to another embodiment, the chemotherapeutic agent is selectedfrom the group consisting of topoisomerase inhibitors, spindle poisonvincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel,docetaxel; alkylating agents: mechlorethamine, chlorambucil,cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine;5-fluorouracil, cytarabine, gemcitabin; podophyllotoxins: etoposide,irinotecan, topotecan, dacarbazin; antibiotics: doxorubicin(adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU),lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions:cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen,leuprolide, flutamide, megestrol acetate.

According to yet another aspect the present invention provides a methodfor alleviating or preventing the damage induced by radiation therapycomprising the step of administering to a subject undergoing radiationtherapy a therapeutically effective amount of a quinazolinone derivativecompound having the general formula I:

wherein: n=1-2R₁ at each occurrence is independently a member of the group consistingof hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and loweralkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy;R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof.

According to one currently preferred embodiment the quinazolinonederivative used in the method of preventing radiation damage ishalofuginone.

According to another currently preferred embodiment the administrationof the quinazolinine compositions of the present invention is prior tothe administration of the radiation therapy.

The present invention is explained in greater detail in the description,figures and claims below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of halofuginone on cell cycle of rabbit aorticsmooth muscle cells (SMC).

FIG. 2 shows the effect of halofuginone on cell cycle of U266 cells.

FIG. 3 shows the effect of combination treatment—halofuginone(HF)+Melphalan—on the viability of U266 cells.

FIG. 4 illustrates leg contraction as a model for radiation-inducedfibrosis.

FIG. 5 shows the influence of halofuginone (μg/mouse) on the contractionof irradiated mice legs.

FIG. 6 shows radiation survival curves for two human pancreatic cancercell lines pre-treated with Halofuginone.

DETAILED DESCRIPTION OF THE INVENTION

The combination of treatments with different modes of action in cancertherapy is currently gaining a lot of enthusiasm. Combining differentmodalities or even specific agents with different mechanism of actionand different adverse effects, allows for better efficacy with fewerside effects.

In this context quinazolinone derivatives, preferably halofuginone, arenow disclosed to improve the effect of other anti-tumor agents ortreatments, either through enhancing the effect of the anti-tumortreatment or through the reduction of adverse effects associate with thetreatment.

Unexpectedly, it has been found, as exemplified in detail herein below,that pharmaceutical compositions comprising quinazolinone derivatives,preferably halofuginone, can synergistically enhance the effectivenessof known anti-tumor treatments including, but not limited to, radiationtherapy and chemotherapy.

Hereinafter, the term “anti tumor treatments” refers to any anti tumortreatment approved for use in a subject. The term “radiation therapy”refers to treatment of cancer through ionizing radiation, as is wellknown in the art. The term “chemotherapy” refers to treatment of adisease characterized by abnormal cell proliferation with chemicals ordrugs. The term “immunotherapy” refers to treatment of disease bymodulation of the immune system and/or responses. The term “hormonaltherapy” refers to treatment of a disease characterized by abnormal cellproliferation with different hormones or their inhibitors. The term“genetic therapy ” refers to treatment of disease characterized byabnormal cell proliferation with compositions containing different genesor gene products, including antisense therapy. The term “subject” refersto the human or animal to whom halofuginone is administered.

According to one aspect the present invention provides a method forincreasing the effectiveness of additional known anti tumor treatments,the method comprising the step of co-administering to a subject in needthereof a pharmaceutical composition comprising as an active ingredienta quinazolinone derivative compound having the general formula I:

wherein: n=1 -2R₁ is at each occurrence independently a member of the group consistingof hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and loweralkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy;R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof,and at least one additional known anti tumor treatment.

According to one currently preferred embodiment, the quinazolinonederivative is halofuginone.

Hereinafter, the term “halofuginone” is defined as a compound having theformula:

and pharmaceutically acceptable salts thereof.

A composition comprising halofuginone preferably further comprises apharmaceutically acceptable carrier for the compound.

According to one embodiment, the known anti tumor treatments applied incombination with the quinazolinone compositions of the present inventionis selected from the group consisting of radiation therapy,chemotherapy, immunotherapy, hormonal therapy and genetic therapy.

According to one currently preferred embodiment the anti tumor treatmentis selected from the group consisting of radiation or chemotherapy.

Some inventors of the present invention have previously disclosed (U.S.Pat. No. 6,420,371 to Pines et al) that halofuginone by itself inhibitstumor progression in vivo. It was suggested that halofuginone mode ofaction in inhibiting tumor progression may be via inhibitingangiogenesis or via substantially inhibiting deposition of extracellularcell matrix components, or via a combination of both.

The mechanism by which halofuginone enhances the efficacy ofchemotherapy or irradiation in treatments of tumor cells is not clear.Without wishing to be bound to a specific mechanism, halofuginone mayact by increasing the sensitivity of tumor cells to the toxic effects ofionizing radiation or chemotherapy treatment, although other mechanismscan also be involved.

Some of the most effective and commonly used chemotherapy agents,including but not limited to taxol, gemacetabin, vinca alkaloids andmany others, are known to affect cancer cells in a specific stage of thecell cycle. These agents may therefore be described as “cell cyclespecific agents”. The cell cycle can be described as a sequence ofphases through which the cell proceeds as it proliferates. The phases ofthis cycle are denoted G1, S, G2 and M, where G1 is the gap precedingsynthesis of DNA, S is the phase during which the cell synthesizes DNA,G2 is the gap between the S phase and division or mitosis (M). Cellsthat are not proliferating may be arrested in a stage referred to as G₀.

It was shown by one of the inventors of the present invention thathalofuginone reversibly arrests cells in the G₀/G₁ stage. Upon removalof halofuginone, cells are able to enter the S phase and continuecycling (Nagler et. al. Kidney Int. Vol. 52(1997), pp. 1561-1569).Therefore, the co-administration of halofuginone as a synchronizingagent will sensitize the tumor cells towards a cell cycle specificagent, as defined above. Upon exposure to halofuginone the cell cyclewill be arrested, whereas upon its removal the cancer cells will regaintheir normal cycling. Effectively, this serves to synchronize the cells,thus bringing a larger proportion of the cells to the specific stage ofthe cell cycle where they will be sensitive to the effects of thechemotherapeutic agent.

According to one embodiment of the present invention the enhancement ofthe effectiveness of known anti-tumor treatments is obtained bypretreatment with a quinazolinone of general formula I, preferablyhalofuginone. This is particularly effective when the additionalanti-tumor treatment is selected from the group consisting of radiationtherapy and chemotherapy.

According to yet another embodiment of the present invention theenhancement of the effectiveness of known anti-tumor treatments isobtained by treatment with a quinazolinone of general formula I,preferably halofuginone, at substantially the same time as the treatmentwith the additional known anti-tumor treatment. Administration may be ina single composition or in separate compositions as appropriate for theoptimal formulation of each agent.

According to another aspect the present invention provides a combinedcomposition for increasing the effectiveness of known anti-tumortreatments comprising a quinazolinone derivative compound having thegeneral formula I:

wherein: n=1-2R₁ is at each occurrence independently a member of the group consistingof hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and loweralkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy;R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof,further comprising at least one additional anti tumor agent.

According to one currently preferred embodiment of the presentinvention, the combined composition comprises halofuginone.

Specific non-limiting examples of chemotherapeutic agents that arebeneficially administered together with quinazolinone derivativesaccording to the present invention include, but are not limited to,doxorubicin, daunorubicin, idarubicin, epirubicin, melphalan,dacarbazine, cisplatin, carboplatin and mitomycin.

Additional cancer chemotherapeutic agents suitable for use incombination with the compositions and methods of the present inventionmay be selected from the following categories: topoisomerase inhibitors,spindle poison vincas: vinblastine, vincristine, vinorelbine (taxol),paclitaxel, docetaxel; alkylating agents: mechlorethamine, chlorambucil,cyclophosphamide, ifosfamide; methotrexate; 6-mercaptopurine;5-fluorouracil, cytarabine, gemcitabin; podophyllotoxins: etoposide,irinotecan, topotecan, dacarbazin; antibiotics: bleomycin; nitrosoureas:carmustine (BCNU), lomustine; inorganic ions: cisplatin, carboplatin;interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide,megestrol acetate.

According to yet another aspect the present invention provides the useof a quinazolinone derivative having the general formula I:

wherein: n=1-2R₁ at each occurrence is independently a member of the group consistingof hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and loweralkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy; andR₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof,in preparation of a medicament for treating a tumor in combinationtherapy with at least one additional known anti tumor treatment, forimproving the effectiveness of the anti tumor treatment.

According to one preferred embodiment, the quinazolinone derivative usedin combination therapy is halofuginone.

According to one embodiment, the combined therapy comprises anadditional known anti tumor treatment selected from the group consistingof radiation therapy, chemotherapy, immunotherapy, hormonal therapy andgenetic therapy.

According to one currently preferred embodiment the combined therapycomprises an additional known anti tumor treatment selected from thegroup consisting of radiation therapy or chemotherapy.

According to another embodiment, the chemotherapeutic agent is selectedfrom the group consisting of topoisomerase inhibitors, spindle poisonvincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel,docetaxel; alkylating agents: mechlorethamine, chlorambucil,cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine;5-fluorouracil, cytarabine, gemcitabin; podophyllotoxins: etoposide,irinotecan, topotecan, dacarbazin; antibiotics: doxorubicin(adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU),lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions:cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen,leuprolide, flutamide, megestrol acetate.

According to another aspect the present invention provides a method foralleviating or preventing the damage induced by radiation therapy,comprising the step of administering to a subject undergoing radiationtherapy a therapeutically effective amount of quinazolinone derivativecompounds having the general formula I:

wherein: n=1-2R₁ which at each occurrence is independently a member of the groupconsisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyl andlower alkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy;R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof.

According to one currently preferred embodiment, the quinazolinonecompound is halofuginone.

According to one currently preferred embodiment, the quinazolinonecomposition of the present invention is administered prior toadministration of the radiation therapy.

According to one embodiment the compositions of the present inventionmay be administered orally or parenterally.

Pharmaceutical compositions for oral administration are formulated asaqueous or solid dosage form.

According to one embodiment the pharmaceutical compositions for oraladministration are formulated in aqueous form selected from the groupconsisting of sterile solutions, sterile suspensions, sterile drysoluble lyophilized powders ready for reconstitution by combination witha vehicle just prior to use, sterile emulsions, microemulsions,dispersions, liposomal dosage forms, lipid complexes such as withcholesterol derivatives and phospholipids.

According to one embodiment the solutions and vehicles are selected fromthe group consisting of aqueous or non-aqueous solutions.

According to one embodiment, the aqueous oral vehicle may furthercomprise cosolvents such as ethyl alcohol, polyethylene glycol,propylene glycol and mixture thereof.

According to one embodiment the sterile formulation may compriselyophilized powders ready for reconstitution by aqueous vehicle.

Optionally, at least one additional ingredient selected from the groupconsisting of, preservatives, antioxidants and tonicity controllingagents can be used.

According to one embodiment the preservatives are selected from thegroup consisting of benzyl alcohol, methyl paraben, propyl paraben,sodium salts of methyl paraben.

According to one embodiment the tonicity controlling agents are selectedfrom the group comprising of sodium chloride, mannitol, dextrose,glucose, lactose and sucrose.

According to yet another embodiment the pharmaceutical compositions fororal administration are formulated in a solid form selected from thegroup consisting of tablets, capsules, sachets, powders, granules andlozenges.

According to one embodiment the present invention relates to a solidpharmaceutical formulated as tablets containing in addition to theactive compound suitable excipients include, but are not limited to,starches, gum arabic, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include lubricating agents such as, forexample, talc, magnesium stearate and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropyl hydroxybenzoates; sweetening agents; or flavoring agents.Polyols, buffers, and inert fillers may also be used. Examples ofpolyols include, but are not limited to: mannitol, sorbitol, xylitol,sucrose, maltose, glucose, lactose, dextrose, and the like. Suitablebuffers encompass, but are not limited to, phosphate, citrate,tartarate, succinate, and the like. Other inert fillers which may beused encompass those which are known in the art and are useful in themanufacture of various dosage forms. If desired, the solidpharmaceutical compositions may include other components such as bulkingagents and/or granulating agents, and the like. The compositions of theinvention can be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art.

Pharmaceutical compositions for parenteral administration are formulatedfor intravenous injections, intravenous infusion, intradermal,intralesional, intramuscular, and subcutaneous injections or depots; orthey may be administered parenterally by means other than an injection,for example, it could be introduced laparascopically, intravesicularly,or via any orifice not related to the gastrointestinal tract.

According to one embodiment the pharmaceutical compositions forparenteral administration are preferably a formulation selected from thegroup consisting of sterile solutions ready for injection, sterilesuspensions ready for injection, sterile dry soluble lyophilized powdersready for reconstitution by combination with a vehicle just prior touse, sterile emulsions, microemulsions, dispersions, liposomal dosageforms, lipid complexes such as with cholesterol derivatives andphospholipids.

According to one embodiment the solutions and vehicles are selected fromthe group consisting of aqueous or non-aqueous solutions. In a preferredembodiment the aqueous parenteral solutions and vehicles are selectedfrom the group consisting of sterile water for injection, sodiumchloride injection, Ringers injection, isotonic dextrose injection,dextrose and lactated Ringers injection.

According to one embodiment, the aqueous parenteral vehicle may furthercomprise cosolvents also referred to as water miscible solvents such asethyl alcohol, polyethylene glycol, propylene glycol and mixturethereof.

According to one embodiment the sterile injection may compriselyophilized powders ready for reconstitution by aqueous vehicle. Suchlyophilized powders comprising quinazolinone derivative and a solidpharmaceutically acceptable buffering agent or a water-soluble organicacid. The buffering agents or organic acids used in the composition maybe any non-toxic buffering agent or organic acid approved for parenteraluse.

Optionally, at least one additional ingredient selected from the groupconsisting of, preservatives, antioxidants and tonicity controllingagents can be used.

According to one embodiment the preservatives are selected from thegroup consisting of benzyl alcohol, methyl paraben, propyl paraben,sodium salts of methyl paraben.

According to one embodiment the tonicity controlling agents are selectedfrom the group comprising of sodium chloride, mannitol, dextrose,glucose, lactose and sucrose.

Although the specific quinazolinone derivative “halofuginone” isreferred to throughout the specification, it is understood that otherquinazolinone derivatives may be used in its place, these derivativeshaving the general formula I:

wherein: n=1-2R₁ is at each occurrence independently a member of the group consistingof hydrogen, halogen, nitro, benzo, lower alkyl, phenyl and loweralkoxy;R₂ is a member of the group consisting of hydroxy, acetoxy and loweralkoxy;R₃ is a member of the group consisting of hydrogen and loweralkenoxy-carbonyl; or pharmaceutically acceptable salts thereof.

As previously disclosed by inventors of the present invention (U.S. Pat.No. 6,420,371 to Pines et. al; U.S. patent application Ser. No.09/762715 to Pines et. al), halofuginone activity in vivo cannot alwaysbe predicted from its activity in vitro. Therefore, there is need toexamine the capability of halifuginone to enhance the efficacy of knownanti tumor treatments in an in vivo model, as described in greaterdetails in the Examples below.

While the invention will now be described in connection with certainpreferred embodiments in the following figures and examples so thataspects thereof may be more fully understood and appreciated, it is notintended to limit the invention to these particular embodiments. On thecontrary, it is intended to cover all alternatives, modifications andequivalents as may be included within the scope of the invention asdefined by the appended claims. Thus, the following figures and exampleswhich include preferred embodiments will serve to illustrate thepractice of this invention, it being understood that the particularsshown are by way of example and for purposes of illustrative discussionof preferred embodiments of the present invention only, and arepresented in the cause of providing what is believed to be the mostuseful and readily understood description of formulation procedures aswell as of the principles and conceptual aspects of the invention.

EXAMPLES Example 1 The Effect of Combined Treatment of Halofuginone and1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

Halofuginone was tested in the human T98G glioblastoma xenograftimplanted subcutaneously or intracranially. Mice were implanted with thehuman T98G glioblastoma tumor cells subcutaneously in a thigh orintracranially.

Halofuginone was administered orally by gavage at dose levels of0.1,0.2, and 0.5 mg/kg/day, once per day, on days 4 through 34 days posttumor implantation. Each group contained 5 mice.

The endpoint for the subcutaneous tumor was tumor growth delay while theendpoint for the intracranial tumor was increase-in-lifespan (survival).

There was no effect of halofuginone on the body weight of the animals.TABLE 1 Response of the Human T98G Glioblastoma Multiforme to treatmentwith BCNU along with halofiginone TUMOR GROWTH DELAY SURVIVAL TREATMENTGROUP Days (sc tumor) Days (ic tumor) CONTROL 0 64 ± 10 No treatmentBCNU (15 mg/kg), ip, 3.9 ± 0.4 97 ± 21 days 7, 9, 11 BCNU + 0.1 mg/kg6.4 ± 0.8 103 ± 29  halofiginone BCNU + 0.2 mg/kg 11.1 ± 1.0  148 ± 4 halofiginone BCNU + 0.5 mg/kg 12.2 ± 1.6  119 ± 21  halofiginone

Combination of halofuginone with BCNU elevated dramatically the tumorgrowth delay from 3.8 days to 12 days.

In the intracranial model halofuginone in combination with BCNUsignificantly increased the life span of mice compared to BCNU alone.The pharmacological optimal dose of 0.2 mg/kg body weight prolonged thesurvival beyond the scope of the study (animals sacrificed in goodhealth after 150 days).

Both effects were dose dependent.

Example 2 Halofuginone Induces Cell Cycle Arrest in Rabbit Aortic SmoothMuscle Cells (SMC)

Experiments were conducted to determine the specific phase of the cellcycle in which SMC treated with halofuginone were arrested.

As determined by [³H]-thymidine incorporation, addition of 10% FBS togrowth-arrested, quiescent SMC promotes entry of the cells to S phaseafter a G₁ period of 16 hours. Maximal DNA synthesis was seen 20 hoursafter serum-stimulation (FIG. 1).

When halofuginone (10⁻⁷ M) was added with 10% FBS, only low levels of[³H]-thymidine incorporation were observed (FIG. 1). It was nextdetermined whether halofuginone arrested proliferation at a specificstage in the cell cycle. For these experiments, quiescent SMCs were keptin 10% FBS plus 10⁻⁷ M halofuginone for 24 hours. The cultures were thenwashed and placed in 10% FBS with [³H]-thymidine and withouthalofuginone. At various times after halofuginone removal, the cellswere harvested and thymidine incorporation was determined. Whenhalofuginone-treated cells were released from growth-arrest, there was alag of 4-6 hours before initiation of DNA synthesis, which peaked by 10hours (FIG. 1).

Since quiescent G₀-arrested SMCs require a minimum of about 16 hours topass through G₀, pretreatment with 10% FBS plus halofuginone permittedcell cycle progression to a point about 4-6 hours from S phase. Thus, inthe continual presence of halofuginone, SMC progress into G₁ andreversibly arrest at late G₁ phase.

Example 3 Halofuginone Arrest Rat Mesangial Cells (RMC) in G₀/G₁ Phase

Further experiments were conducted to determine whether halofuginonearrests mesangial cell proliferation at a specific phase of the cellcycle.

For this purpose sub confluent RMC's were kept in 10% FCS in the absenceor presence of 150 ng/ml halofuginone for 24 hours. The cells were thenharvested, stained with propidium iodide and analyzed by FACScan. Thepercentage of cells progressing into G₂/M phase was reduced byhalofuginone from 20% to 7%. The percentage of cells in G₀/G₁ wasincreased from 38% in the absence of halofuginone to 65% in the presenceof halofuginone. These results indicate that in the presence ofhalofuginone, a large proportion of the mesangial cells are arrested inthe G₀/G₁ phase.

Example 4 Combination Treatment of Halofuginone and Melphalan onMultiple Myeloma Cells

Materials and Methods

Multiple Myeloma (MM) cell line U266B1 was purchased from ATCC(TIB-196).

WST-1 reagent (Roche 1 644 807)

Melphalan-SIGMA M2011

1. WST—Viability Test

Cells were grown in RPMI supplemented with 20% FCS. Cells were seededinto 96 well plates (30K cell/well) with various concentrations ofhalofuginone or melphalan. After incubation period WST reagent was addedto wells and cells were incubated for about 24 hours at 37° C., 5% CO₂.Absorbance was measured at 440 nm using scanning multi-wellspectrophotometer (ELISA reader).

2. Cell Cycle Analysis

Cells (10⁶) were incubated for 48 hours with different concentrations ofhalofuginone. The cells were permeabilized with 70% ethanol in PBS for30 minutes at 4° C., and then incubated with 0.5 ml of a 50 μg/mlPropidium Iodide solution containing 20 U/ml RnaseA for 30 minutes.Cells were analyzed by flow cytometry.

Results

As shown in FIG. 2, treatment of U266 cells with halofuginone causedelevation in the number of cells arrested in G1 phase.

As a second step, the effect of pretreatment with halofuginone on thesensitivity of MM cells to melphalan, a known anti-tumor treatment, wasexamined.

Cells (30K) were treated for 48 hours with 60 nM halofuginone withsubsequent treatment with melphalan for 72 hours in variousconcentrations. At the end of the incubation, cell viability wasmeasured by WST-viability test. Sequential treatment of halofuginone andmelphalan was more effective than treatment with melphalan alone (FIG.3), thus demonstrating the synergistic effect between halofuginone andmelphalan treatments. It is suggested that the synchronization of thecells in pre-G1 phase of the cell cycle rendered them more sensitive tothe melphalan treatment.

Example 5 Halofuginone Decreased Fibrosis Induced by Radiation

Mice were injected intraperitoneally once daily with 1-5 μg/mouse ofhalofuginone, for a period of 4 months.

The right leg only of each animal was radiated with 35Gy or 45Gy. In thecontrol group mice did not receive halofuginone and the right leg wasradiated with 35Gy or 45Gy. Leg contraction was measured as demonstratedin FIG. 4. Measurements were taken within time periods of 2 to 4 monthsafter radiation.

As shown by FIG. 5, dramatic decrease in the “leg length difference”between the right and left leg is observed in halofuginone treatedanimals. The effect of halofuginone can be observed post radiation at 2and 4 months, at 35Gy and 45 Gy radiation and at all of the usedhalofuginone concentrations (1-5μg/mouse).

In general, it can be concluded that halofuginone reduced the radiationeffects in this in-vivo model, as the irradiated leg of mice thatreceived halofuginone was definitely less stiff, and the skin was lessdry in comparison to mice that did not receive halofuginone.

Example 6 Halofuginone Acts as a Radiation Sensitizer

Two pancreatic cancer cell lines: 3602 Xrt and 3602 Zyrd/Xrt wereincubated with or without 250 nM halofuginone for 24 hr, than radiatedwith 0-8 Gy. Survival fraction of the cell was determined. As shown inFIG. 6, halofuginone caused a decrease in the survival fraction ofapproximately 50%. These results confirm the observation thathalofufinone increase the sensitivity of the tumor cells to theanti-tumorigenic treatment of radiation.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed chemical structures andfunctions may take a variety of alternative forms without departing fromthe invention. Thus the expressions “means to . . . ” and “means for . .. ”, or any method step language, as may be found in the specificationabove and/or in the claims below, followed by a functional statement,are intended to define and cover whatever chemical structure, orwhatever function, which may now or in the future exist which carriesout the recited function, whether or not precisely equivalent to theembodiment or embodiments disclosed in the specification above, i.e.,other means or steps for carrying out the same functions can be used;and it is intended that such expressions be given their broadestinterpretation.

1. A method for improving the effectiveness of an anti-tumor treatmentcomprising the step of co-administering to a subject in need thereof apharmaceutical composition comprising as an active ingredient aquinazolinone derivative compound having the general formula I:

wherein: n=1-2 R₁ at each occurrence is independently selected from thegroup consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyland lower alkoxy; R₂ is a member of the group consisting of hydroxy,acetoxy and lower alkoxy; R₃ is a member of the group consisting ofhydrogen and lower alkenoxy-carbonyl; or pharmaceutically acceptablesalts thereof, and at least one additional anti tumor treatment.
 2. Themethod according to claim 1 wherein the subject is human.
 3. The methodaccording to claim 1 wherein the administration of the quinazolinonecomposition is prior to the administration of the at least oneadditional anti-tumor treatment.
 4. The method according to claim 1wherein the administration of the quinazolinone composition issubstantially at the same time as the administration of the at least oneadditional anti-tumor treatment.
 5. The method according to claim 4wherein the co-administration is in a single pharmaceutical composition.6. The method according to claim 4 wherein the co-administration is inseparate pharmaceutical compositions.
 7. The method according to any oneof claims 1-4 wherein the anti tumor treatment is radiation therapy. 8.The method according to any one of claims 1-6 wherein the anti tumortreatment is chemotherapy.
 9. The method according to any one of claims1-6 wherein the anti tumor treatment is selected from the groupconsisting of immunotherapy, hormonal therapy and genetic therapy. 10.The method according to claim 1 wherein the improvement in effectivenessis achieved by enhancement of cellular sensitivity to the anti tumortreatment.
 11. The method according to any one of claims 1-10 whereinthe compound of formula I is halofuginone or a pharmaceuticallyacceptable salt, solvent or hydrate thereof.
 12. The method according toclaim 8, wherein the additional agent used for chemotherapy is selectedfrom the group consisting of topoisomerase inhibitors, spindle poisonvincas: vinblastine, vincristine, vinorelbine (taxol), paclitaxel,docetaxel; alkylating agents: mechlorethamine, chlorambucil,cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine;5-fluorouracil, cytarabine, gemcitabin; podophyllotoxins: etoposide,irinotecan, topotecan, dacarbazin; antibiotics: doxorubicin(adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU),lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions:cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen,leuprolide, flutamide, megestrol acetate.
 13. A combined pharmaceuticalcomposition comprising as an active ingredient a quinazolinonederivative compound having the general formula I:

wherein: n=1-2 R₁ at each occurrence is independently selected from thegroup consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyland lower alkoxy; R₂ is a member of the group consisting of hydroxy,acetoxy and lower alkoxy; R₃ is a member of the group consisting ofhydrogen and lower alkenoxy-carbonyl; or pharmaceutically acceptablesalts thereof, and at least one pharmaceutically acceptable carrier ordiluents; further comprising at least one additional anti tumor agent.14. The pharmaceutical composition according to claims 13 wherein thecompound of formula I is halofuginone or a pharmaceutically acceptablesalt, solvent or hydrate thereof.
 15. The pharmaceutical compositionaccording to claim 13 wherein the anti tumor agent is a chemotherapeuticagent.
 16. The pharmaceutical composition according to claims 15,wherein the chemotherapeutic agent is selected from the group consistingof topoisomerase inhibitors, spindle poison vincas: vinblastine,vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylatingagents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan,ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine,gemcitabin; podophyllotoxins: etoposide, irinotecan, topotecan,dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin;nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin,daunorubicin; inorganic ions: cisplatin, carboplatin; interferon,asparaginase; hormones: tamoxifen, leuprolide, flutamide, megestrolacetate.
 17. Use of a quinazolinone derivative compound having thegeneral formula 1:

wherein: n=1-2 R₁ at each occurrence is independently selected from thegroup consisting of the hydrogen, halogen, nitro, benzo, lower alkyl,phenyl and lower alkoxy; R₂ is a member of the group consisting ofhydroxy, acetoxy and lower alkoxy; R₃ is a member of the groupconsisting of hydrogen and lower alkenoxy-carbonyl; or pharmaceuticallyacceptable salts thereof, in the preparation of a medicament fortreating a tumor in combination therapy with at least one additionalanti tumor treatment, thereby improving the effectiveness of the antitumor treatment.
 18. Use according to claim 17, wherein the additionalanti tumor treatment is radiation therapy.
 19. Use according to claim17, wherein the additional anti tumor treatment is chemotherapy.
 20. Useaccording to claim 17, wherein the additional anti tumor treatment isselected from the group consisting of immunotherapy, hormonal therapyand genetic therapy.
 21. Use according to claim 17, wherein theimprovement is achieved by enhancement of cellular sensitivity to theanti tumor treatment.
 22. Use according to claim 17 wherein the compoundof formula I is halofuginone or a pharmaceutically acceptable salt,solvent or hydrate thereof.
 23. Use according to claim 19 wherein thechemotherapeutic agent is selected from the group consisting oftopoisomerase inhibitors, spindle poison vincas: vinblastine,vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylatingagents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan,ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine,gemcitabin; podophyllotoxins: etoposide, irinotecan, topotecan,dacarbazin; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin;nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin,daunorubicin; inorganic ions: cisplatin, carboplatin; interferon,asparaginase; hormones: tamoxifen, leuprolide, flutamide, megestrolacetate.
 24. The pharmaceutical composition of any one of claims 13-16formulated in a form suitable for administration of the compositionorally or parenterally.
 25. The pharmaceutical composition according toclaims 24 wherein the formulation for parenteral administration isselected from a dosage form suitable for intravenous injections,intravenous infusion; intradermal, intralesional, intramuscular, andsubcutaneous injections or depots; for administration parenterally bymeans other than injection, laparascopically, intravesicularly, orintralesionally.
 26. The pharmaceutical composition according to claim24 formulated for oral administration in a form selected from a powder,granules, suspensions or solutions in water or non aqueous media,sachets, capsules or tablets.
 27. A method for alleviating or preventingthe damage induced by radiation therapy comprising the step ofadministering to a subject undergoing radiation therapy a pharmaceuticalcomposition comprising as an active ingredient a quinazolinonederivative compound having the formula I:

wherein: n=1-2 R₁ at each occurrence is independently a member of thegroup consisting of hydrogen, halogen, nitro, benzo, lower alkyl, phenyland lower alkoxy; R₂ is a member of the group consisting of hydroxy,acetoxy and lower alkoxy; R₃ is a member of the group consisting ofhydrogen and lower alkenoxy-carbonyl; or pharmaceutically acceptablesalts thereof, further comprising a pharmaceutically acceptable carrier.28. The method according to claim 27 wherein the compound according toformula I is halofuginone a pharmaceutically acceptable salt, solvent orhydrate thereof.
 29. The method according to claim 27 wherein theadministration is prior to the administration of radiation therapy.